Tuberculosis or "TB" is an acute or chronic infection caused by Mycobacterium tuberculosis. Tuberculosis continues to be a major health concern in both the United States and abroad. According to the World Health Organization's estimates, 1.7 billion people (one-third of the world population) harbor tuberculosis bacteria in their bodies and approximately 3 million of these people die each year from tuberculosis infection. See, Kaufman, et al., Trends in Microbiology 1:2-5 (1993). This means that tuberculosis is responsible for 6% of the total global mortality. See, Kaufman, id.
Mycobacterium tuberculosis is a member of the genus mycobacterium. Pathogenic Mycobacteria are mostly slow growing organisms which are shaped like straight or slightly curved rods and are sometimes branching or filamentous. Mycobacteria are sometimes referred to as acid-fast bacilli (AFB) because application of alcohol (e.g., acid-alcohol or 95% ethanol with 3% hydrochloric acid) to mycobacteria stained with basic dye will not decolorize them. Typically, mycobacteria are obligate aerobes and can be characterized as gram-positive.
Mycolic acids are major constituents of the mycobacterial cell wall, representing up to 30% of the dried cell mass. They are .alpha.-alkyl, .beta.-hydroxy fatty acids which in mycobacteria range in size from sixty to ninety carbons. Takayama and Qureshi, The Mycobacteria: A Sourcebook, Part A, G.P. Kubica and L.G. Wayne, Eds., Marcel Dekker, Inc., New York, N.Y. (1984). Since their isolation by Stodola, Lesuk and Anderson in 1938, the chemistry and structure of these lipids has become the subject of an extensive literature. See, Goren, Bacteriol Rev. 36, 33 (1972); Minnikin, The Biology of the Mycobacteria, C. Ratledge, and J. Stanford, Eds. (Academic Press, San Diego, Calif., 1982), pp. 95-184.
The genus mycobacterium includes a number of highly pathogenic organisms besides M. tuberculosis. These other pathogenic forms of mycobacteria include: M. leprae, M. avium, M. bovis, M. chelonei (also known as borstelense and abscessus), M. africanum, M. marinium (also known as balnei and platypoecilus, the causative agent of "swimming pool granuloma"), M. buruli (also known as ulcerans), M. fortuitum (also known as giae, minetti, and ranae), M. haemophilum, M. intracellulare, M. kansasii (also known as luciflavum), M. littorale (also known as xenopi), M. malrnoense, M. marianum (also known as scrofulaceum and paraffinicum), M. simiae, M. szulgai, and M. ulcerans (which is the agent responsible for Buruli ulcer). There are also non-pathogenic forms of mycobacteria which include: M. gordonae (also known as aquae), M. gastri, M. phlei (also known as moelleri and as timothy bacillus), M. nonchrornogenicum, M. smegmatis, M. terrae, M. triviale, and M. vaccae.
It has been found that pathogenic and non-pathogenic forms of mycobacteria biosynthesize different mycolic acids. Pathogenic forms of mycobacteria, including M. tuberculosis, M. avium, M. kansasi, M. leprae, M. ulcerans, and M. marinum, uniformly modify their major mycolic acids at two positions by enzymatically transforming a double bond into a cyclopropane ring. Minnikin, The Biology of the Mycobacteria, C. Ratledge, and J. Stanford, Eds. (Academic Press, San Diego, Calif., 1982) pp. 95-184; Daffe et al., Res. Microbiol 142, 397(1991). By contrast, non-pathogenic mycobacteria, such as M. smegrnatis, do not cyclopropanate their mycolic acids. This difference is illustrated in the proposed mycolic acid biosynthesis shown in FIG. 1 for the pathogenic mycobacterium M. tuberculosis and the non-pathogenic mycobacterium M. smegmatis.
Conventional therapy for tuberculosis includes treatment with such pharmaceuticals as isoniazid ("INH"), ethambutol, streptomycin, rifampin, rifabutin, clarithromycin, ciprofloxacin, clofazamine, azithromycin, ethionamide, pyrazinamide, amikacin and/or resorcinomycin A. A new therapeutic for tuberculosis is described in the inventors' co-pending application Ser. No. 08/210,519, the disclosures of which are incorporated by reference. In many cases, the initial treatment for tuberculosis includes INH in combination with at least one other drug, such as ethambutol, streptomycin, rifampin or ethionamide. While treatment of tuberculosis patients with drug therapies involving INH is often effective, use of INH can have serious drawbacks. For example, treatment with INH often causes severe, sometimes fatal, hepatitis. Also, INH causes peripheral neuropathy and liver dysfunction in some recipients. Moreover, there are emerging strains of Mycobacterium tuberculosis which are resistant to multiple existing drug treatments, particularly INH. See, D.E. Snider and W.L. Roper, N. Engl. J. Med. 326, 703 (1992). These new mutant strains of Mycobacterium tuberculosis will present grave public health risks in the years ahead unless new and more effective treatments are devised to combat them.
A large proportion of the current arsenal of chemotherapeutics against Mycobacterium tuberculosis and other pathogenic forms of mycobacteria are thought to affect the biosynthesis of the cell-wall components of such mycobacteria, particularly the mycolic acids. This biosynthetic pathway is thought to be the target of INH, ethionamide, thiocarlide and possibly ethambutol. See, A. Banerjee et al., Science, 263:227 (1994); F. Winder, The Biology of The Mycobacteria, Vol. 1, C. Rutledge, J. Stanford, Eds., Academic Press, San Diego, Calif. (1982). In spite of the importance of this biosynthetic pathway as a chemotherapeutic target, the inventors do not know of a single enzyme activity or gene directly involved in the biosynthesis of mycolic acids which has previously been identified in the art. Even the recent identification of the inhA gene by Banerjee et al. appears to relate to mycolate transport or an early component of fatty acid biosynthesis rather than a specific component of the mycolate biosynthetic pathway. A. Banerjee et al., id.
What is greatly needed in the art is the identification, isolation and purification of genes and enzymes involved in the biosynthesis of mycolic acids in pathogenic mycobacteria, particularly those genes and enzymes likely to be found in the new drug resistant strains of Mycobacterium tuberculosis. From such identification, isolation and purification, new therapeutics can be developed and tested. To the extent such genes and enzymes are common to all forms of pathogenic mycobacterium, there is reason to believe that new therapeutics will be effective against the new mutant strains of Mycobacterium tuberculosis.